—Parasympathetic stimulation of the heart acts through M₂-muscarinic acetylcholine receptors to regulate ion channel activity and subsequent inotropic status. Although muscarinic signal transduction is mediated via pertussis toxin-sensitive G proteins Gα_i/o, the specific signal transduction requirements of Gα_i2 and Gα_i3 in mediating muscarinic regulated L-type calcium currents (I_{Ca, L}), intracellular calcium, and cell contractility remain to be determined. Adult ventricular myocytes were isolated from Gα_i2-null mice, Gα_i3-null mice, and their wild-type littermates. Cell shortening, intracellular calcium levels, and I_{Ca, L} were all measured in response to isoproterenol, a β-adrenergic receptor agonist, and carbachol, a cholinergic receptor agonist. With isoproterenol stimulation, myocytes from all groups demonstrated a marked increase in calcium currents, correlating with augmented intracellular calcium transient amplitude and cell shortening. Carbachol significantly attenuated the isoproterenol response in wild-type and Gα_i3-null cells but had no effect in Gα_i2-null cells. This study demonstrates that Gα_i2, but not Gα_i3, is required for muscarinic inhibition of the β-adrenergic response in adult murine ventricular myocytes.